Adenocarcinoma of the esophagus and gastroesophageal junction is rising faster than any other cancer in the United States. In order to arrest this trend, advances in early detection must be made. We hypothesize that unique molecular alterations occurring in premalignant and malignant esophageal and gastric epithelia will constitute biomarkers for earlier diagnosis and improved prognostication to direct screening, prevention, and treatment efforts. The studies proposed herein will identify molecular signatures of early and late esophagogastric adenocarcinogenesis. Aim number 1: To generate pure cellular populations from premalignant and frankly malignant primary human gastroesophageal lesions. Number 1a: Xenografts will be generated in nude mice from human esophageal and gastric adenocarcinomas. Number 1b: Pure populations of metaplastic and dysplastic Barrett s esophagus, intestinal metaplasia of the stomach, and primary gastric and esophageal adenocarcinomas will be produced using laser capture microdissection (LCM). Aim number 2: We will discover peptide biomarkers by employing a novel peptide sequencing methodology which utilizes tandem mass spectrometry. Peptide sequences found to be differentially expressed in tumors will be subsequently explored for known homology in protein databases. Antibodies to known and novel potentially secreted or cancer-related proteins will be obtained or generated and used for immunohistochemical analyses, Western blots, and to probe primary tissue microarrays containing all neoplastic stages. Aim number 3: We will identify genes whose expression levels are increased or decreased at each stage of esophageal or gastric carcinogenesis. Number 3a: We will utilize serial analysis of gene expression (SAGE) to generate and contrast global gene expression profiles. Number 3b: We will derive transcriptomes of each stage of esophageal or gastric neoplasia using large, comprehensive screening arrays created on nylon membranes in pilot studies, and then we will create multiple small signature arrays on glass slides, based on peptide studies, SAGE, and large nylon array data. These smaller signature arrays will be tested on our large bank of tissues and prospectively on a larger population of patients possessing all stages of esophagogastric neoplastic progression. Aim number 4: To identify signature allelic loss patterns (allelotypes) at each stage of esophagogastric neoplastic progression using novel automated technology. Aim number 5: To determine the complete sets of coding region microsatellite mutations (instabilotypes) of early and advanced esophageal and gastric neoplastic lesions. Aim number 6: To determine the clinical significance of biomarkers by correlating molecular findings with corresponding initial and followup clinicopathologic data, using appropriate statistical techniques.